The Description of Pseudoalteromonas apostichopi sp. nov., Vibrio apostichopi sp. nov., and Marinobacter apostichopi sp. nov. from the Fertilized Eggs and Larvae of Apostichopus japonicus.

Three novel bacterial strains, FE4T, FE10T, and LA51T, which are phylogenetically affiliated to the genera Pseudoalteromonas, Vibrio, or Marinobacter, respectively, isolated from fertilized eggs and juveniles of sea cucumber Apostichopus japonicus were characterized by a genome-based taxonomical approach including multilocus sequence analysis (MLSA) combined with classical phenotypic and chemotaxonomic characterizations. A molecular network reconstructed on the basis of nucleotide sequences of four phylogenetic maker protein genes revealed that the strains FE4T, FE10T, and LA51T were closely related to Pseudoalteromonas shioyasakiensis, Vibrio lentus, and Marinobacter similis, respectively. Average nucleotide identity (ANI) comparisons against phylogenetically related species to FE4T, FE10T, and LA51T demonstrated that each newly described strain could not be identified as any previously described species within each genus showing < 95% ANI: 91.3% of FE4T against P. shioyasakiensis JCM 18891 T, 92.6% of FE10T against "V. bathopelagicus" Sal10, and 92.6% of LA51T against M. similis A3d10T, in maximum, respectively. Here, we show molecular phylogenetic, genomic, phenotypic, and chemotaxonomic features of the newly described species FE4T, FE10T, and LA51T. We also propose Pseudoalteromonas apostichopi sp. nov. with FE4T (JCM 36173 T = LMG 33143 T) as the type strain, Vibrio apostichopi sp. nov. with FE10T (JCM 36174 T = LMG 33144 T) as the type strain, and Marinobacter apostichopi sp. nov. with LA51T (JCM 36175 T = LMG 33145 T) as the type strain.

A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA-DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1-2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7-8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas. In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

Genomic insights into Pseudoalteromonas sp. JSTW coping with petroleum-heavy metals combined pollution.

Worldwide marine compound contamination by petroleum products and heavy metals is a burgeoning environmental concern. Pseudoalteromonas, prevalently distributed in marine environment, has been proven to degrade petroleum and plays an essential role in the fate of oil pollution under the combined pollution. Nevertheless, the research on the reference genes is still incomplete. Therefore, this study aims to thoroughly investigate the reference genes represented by Pseudoalteromonas sp. JSTW via whole-genome sequencing. Next-generation sequencing technology unfolded a genome of 4,026,258 bp, database including Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were utilized to annotate the genes and metabolic pathways conferring to petroleum hydrocarbon degradation. The results show that common alkane and aromatic hydrocarbon degradation genes (alkB, ligB, yqhD, and ladA), chemotaxis gene (MCP, cheA, cheB, pcaY, and pcaR), heavy-metal resistance, and biofilm genes (σ54, merC, pcoA, copB, etc.) were observed in whole-genome sequence (WGS) of JSTW, which indicated that strain JSTW could potentially cope with combined pollution. The degradation efficiency of naphthalene in 60 h by JSTW was 99% without Cu2+ and 67% with 400 mg L-1 Cu2+ . Comparative genome analysis revealed that genomes of Pseudoalteromonas lipolytica strain LEMB 39 and Pseudoalteromonas donghaensis strain HJ51 shared similarity with strain JSTW, suggesting they are also the potential degradater of petroleum hydrocarbons under combined pollution. Therefore, this study provides a WGS annotation and reveals the mechanism of response to combined pollution of Pseudoalteromonas sp. JSTW.

Identification and analysis of the biological activity of the new strain of Pseudoalteromonas piscicida isolated from the hemal fluid of the bivalve Modiolus kurilensis (F. R. Bernard, 1983).

A cultivated form of bacteria (strain 2202) was isolated from the hemal fluid of the bivalve mollusk Modiolus kurilensis. Based on the set of data collected by genetic and physiological/biochemical analyses, the strain was identified as the species Pseudoalteromonas piscicida. Strain 2202 exhibits antimicrobial activity against Staphylococcus aureus, Candida albicans, and Bacillus subtilis but not against Escherichia coli and Pseudomonas aeruginosa. These activities characterize the behavior of strain 2202 as predator-like and classify it as a facultative predator. Being part of the normal microflora in the hemolymph of M. kurilensis, when external conditions change, strain 2202 shows features of opportunistic microflora. The strain 2202 exhibits selective toxicity towards larvae of various invertebrates: it impairs the early development of Mytilus edulis, but not of Strongylocentrotus nudus. Thus, the selective manner in which P. piscicida strains interact with various species of microorganisms and eukaryotes should be taken into consideration when using their biotechnological potential as a probiotic in aquaculture, source of antimicrobial substances, and factors that prevent fouling.

Population structure, activity potential and ecotype partitioning of Pseudoalteromonas along the vertical water column of the New Britain Trench.

Microbial degradation of organic matter along the vertical profile of the water column is a major process driving the carbon cycle in the ocean. Pseudoalteromonas has been identified as a dominant genus in pelagic marine environments worldwide, playing important roles in the remineralization of organic carbon. However, the current understanding of Pseudoalteromonas was mainly based on shallow water populations or cultivated species. This study analyzed for the first time the structure, activity potential and ecotypes differentiation of Pseudoalteromonas in the water column of the New Britain Trench (NBT) down to 6000 m. Analysis on diversities of the 16S rRNA gene and their transcripts showed that Pseudoalteromonas was greatly enriched in deep-sea waters and showed high activity potentials. The deep-sea Pseudoalteromonas were significantly different from their shallow-water counterparts, suggesting an obvious ecotype division along with the vertical profile. Phylogenetic analysis on the 16S rRNA gene and hsp60 gene of 219 Pseudoalteromonas strains isolated from different depths further showed that the vertical ecotype division could even occur at the strain level, which might be a result of long-term adaptation to environmental conditions at different depths. The discovered depth-specific strains provide valuable models for further studies on adaptation, evolution and functions of the deep-sea Pseudoalteromonas.

Conserved Pigment Profiles in Phylogenetically Diverse Symbiotic Bacteria Associated with the Corals Montastraea cavernosa and Mussismilia braziliensis.

Pigmented bacterial symbionts play major roles in the health of coral holobionts. However, there is scarce knowledge on the diversity of these microbes for several coral species. To gain further insights into holobiont health, pigmented bacterial isolates of Fabibacter pacificus (Bacteroidetes; n = 4), Paracoccus marcusii (Alphaproteobacteria; n = 1), and Pseudoalteromonas shioyasakiensis (Gammaproteobacteria; n = 1) were obtained from the corals Mussismilia braziliensis and Montastraea cavernosa in Abrolhos Bank, Brazil. Cultures of these bacterial symbionts produced strong antioxidant activity (catalase, peroxidase, and oxidase). To explore these bacterial isolates further, we identified their major pigments by HPLC and mass spectrometry. The six phylogenetically diverse symbionts had similar pigment patterns and produced myxol and keto-carotene. In addition, similar carotenoid gene clusters were confirmed in the whole genome sequences of these symbionts, which reinforce their antioxidant potential. This study highlights the possible roles of bacterial symbionts in Montastraea and Mussismilia holobionts.

Pseudoalteromonas caenipelagi sp. nov., isolated from a tidal flat.

A Gram-stain-negative, aerobic, non-spore-forming, motile by single polar flagellum and ovoid or rod-shaped bacterial strain, designated JBTF-M23T, was isolated from tidal flat sediment collected from the Yellow Sea, Republic of Korea. Neighbour-joining phylogenetic tree of 16S rRNA gene sequences showed that strain JBTF-M23T fell within the clade comprising the type strains of Pseudoalteromonas species, clustering with the type strains of P. byunsanensis and P. amylolytica. Strain JBTF-M23T exhibited the highest 16S rRNA gene sequence similarity value (98.6 %) to the type strain of P. rubra and sequence similarities of 98.3 and 97.7 % to the type strains of P. byunsanensis and P. amylolytica, respectively. The DNA G+C content of strain JBTF-M23T from genomic sequence data was 41.98 %. The ANI and dDDH values between strain JBTF-M23T and the type strains of P. rubra, P. byunsanensis and P. amylolytica were 71.3-76.6 and 19.4-19.9 %, respectively. Strain JBTF-M23T contained Q-8 as the predominant ubiquinone and C16 : 1 ω7c and/or C16 : 1 ω6c, C16 : 0 and C18 : 1 ω7c as the major fatty acids. The major polar lipids of strain JBTF-M23T were phosphatidylethanolamine and one unidentified aminolipid. Distinguished phenotypic properties, along with the phylogenetic and genetic distinctiveness, revealed that strain JBTF-M23T is separated from recognized Pseudoalteromonas species. On the basis of the data presented, strain JBTF-M23Tis considered to represent a novel species of the genus Pseudoalteromonas, for which the name Pseudoalteromonas caenipelagi sp. nov. is proposed. The type strain is JBTF-M23T(=KACC 19900T=NBRC 113647T).

Terabase-scale metagenome coassembly with MetaHipMer.

Metagenome sequence datasets can contain terabytes of reads, too many to be coassembled together on a single shared-memory computer; consequently, they have only been assembled sample by sample (multiassembly) and combining the results is challenging. We can now perform coassembly of the largest datasets using MetaHipMer, a metagenome assembler designed to run on supercomputers and large clusters of compute nodes. We have reported on the implementation of MetaHipMer previously; in this paper we focus on analyzing the impact of very large coassembly. In particular, we show that coassembly recovers a larger genome fraction than multiassembly and enables the discovery of more complete genomes, with lower error rates, whereas multiassembly recovers more dominant strain variation. Being able to coassemble a large dataset does not preclude one from multiassembly; rather, having a fast, scalable metagenome assembler enables a user to more easily perform coassembly and multiassembly, and assemble both abundant, high strain variation genomes, and low-abundance, rare genomes. We present several assemblies of terabyte datasets that could never be coassembled before, demonstrating MetaHipMer's scaling power. MetaHipMer is available for public use under an open source license and all datasets used in the paper are available for public download.

Pseudoalteromonas rhizosphaerae sp. nov., a novel plant growth-promoting bacterium with potential use in phytoremediation.

Strain RA15T was isolated from the rhizosphere of the halophyte plant Arthrocnemum macrostachyum growing in the Odiel marshes (Huelva, Spain). RA15T cells were Gram stain-negative, non-spore-forming, aerobic rods and formed cream-coloured, opaque, mucoid, viscous, convex, irregular colonies with an undulate margin. Optimal growth conditions were observed on tryptic soy agar (TSA) plates supplemented with 2.5 % NaCl (w/v) at pH 7.0 and 28 °C, although it was able to grow at 4-32 °C and at pH values of 5.0-9.0. The NaCl tolerance range was from 0 to 15 %. The major respiratory quinone was Q8 but Q9 was also present. The most abundant fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), C17 : 1 ω8c and C16 : 0. The polar lipids profile comprised phosphatidylglycerol and phosphatidylethanolamine as the most abundant representatives. Phylogenetic analyses confirmed the well-supported affiliation of strain RA15T within the genus Pseudoalteromonas, close to the type strains of Pseudoalteromonas neustonica, Pseudoalteromonas prydzensis and Pseudoalteromonas mariniglutinosa. Results of comparative phylogenetic and phenotypic studies between strain RA15T and its closest related species suggest that RA15T could be a new representative of the genus Pseudoalteromonas, for which the name Pseudoalteromonas rhizosphaerae sp. nov. is proposed. The type strain is RA15T (=CECT 9079T=LMG 29860T). The whole genome has 5.3 Mb and the G+C content is 40.4 mol%.

Highly Efficient Capture of Marine Microbial Strains in Seawater Using Bare Fe3O4 Magnetic Beads.

We develop a method to capture marine bacterial strains at high efficiency to replace the conventional two-step collecting method. Lab-made, Fe3O4 magnetic beads were used to firstly verify the feasibility of capture in artificial seawater, using Bacillus velezensis. Almost 100% of the bacteria could be captured and separated within 10 min. Then, the salinity of capture medium was proved to have the most marked effect on the capture process. After that, the broad application and high efficiency of capture were verified using four different bacterial strains from the Pacific Ocean. Subsequently, through adjusting the salinity, the capture efficiency for Pseudoalteromonas sp. and Halomonas meridiana was increased from 20 to ~ 80% in a seawater system, which was used to simulate the in-situ capture conditions. Finally, mixed strains in seawater were successfully captured, and their genomic DNAs were isolated and analyzed. Bare Fe3O4 magnetic beads were initially applied to capture marine microorganisms and this method is convenient and highly efficient and thus has great potential to replace the conventional two-step method.

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5.

Alginate extracted from widely cultured brown seaweed can be hydrolyzed by alginate lyase to produce alginate oligosaccharides (AOS) with intriguing biological activities. Herein, a novel alginate lyase Aly1281 was cloned from marine bacterium Pseudoalteromonas carrageenovora ASY5 isolated from mangrove soil and found to belong to polysaccharide lyase family 7. Aly1281 exhibited maximum activity at pH 8.0 and 50 °C and have broad substrate specificity for polyguluronate and polymannuronate. Compared with other alginate lyases, Aly1281 exhibited high degradation specificity and mainly produced di-alginate oligosaccharides which displayed good antioxidant function to reduce ferric and scavenge radicals such as hydroxyl, ABTS+ and DPPH. Moreover, the catalytic activity and kinetic performance of Aly1281 were highly improved with the addition of salt, demonstrating a salt-activation property. A putative conformational structural feature of Aly1281 was found by MD simulation analysis for understanding the salt-activation effect.

Quorum Quenching Enzyme APTM01, an Acylhomoserine-Lactone Acylase from Marine Bacterium of Pseudoalteromonas tetraodonis Strain MQS005.

Quorum sensing is a system of stimuli and response correlated to population density and involves in pathogen infection, colonization, and pathogenesis. Quorum quenching enzymes as quorum sensing inhibitors have been identified in a number of bacteria and been used to control by triggering the pathogenic phenotype. The marine bacteria of Pseudoalteromonas had wide activity of degrading AHLs as a type of signal molecule associated with quorum sensing. We screened many Pseudoalteromonas strains in large scale to explore genes of quorum quenching enzymes from the China seas by whole-genome sequencing rather than genomic library construction. Nine target strains were obtained and an acylases gene APTM01 from the strain MQS005 belonging to PvdQ type on sub-branch in phylogenetic tree. And the heterogenous host containing the vector with target gene could degrade C10-HSL, C12-HSL and OC12-HSL. The obtained AHL acylase gene would be a candidate quorum quenching gene to apply in some fields. We identified that the strains of Pseudoalteromonas have wide AHL-degrading ability depending on quorum quenching. The strains would be a resource to explore new quorum quenching enzymes.

Functional exploration of Pseudoalteromonas atlantica as a source of hemicellulose-active enzymes: Evidence for a GH8 xylanase with unusual mode of action.

To address the need for efficient enzymes exhibiting novel activities towards cell wall polysaccharides, the bacterium Pseudoalteromonas atlantica was selected based on the presence of potential hemicellulases in its annotated genome. It was grown in the presence or not of hemicelluloses and the culture filtrates were screened towards 42 polysaccharides. P. atlantica showed appreciable diversity of enzymes active towards hemicelluloses from Monocot and Dicot origin, in agreement with its genome annotation. After growth on beechwood glucuronoxylan and fractionation of the secretome, a ß-xylosidase, a α-arabinofuranosidase and an acetylesterase activities were evidenced. A GH8 enzyme obtained in the same growth conditions was further cloned and heterologously overexpressed. It was shown to be a xylanase active on heteroxylans from various sources. The detailed study of its mode of action demonstrated that the oligosaccharides produced carried a long tail of un-substituted xylose residues on the reducing end.

Diversity and distribution of the bmp gene cluster and its Polybrominated products in the genus Pseudoalteromonas.

The production of pentabromopseudilin and related brominated compounds by Pseudoalteromonas spp. has recently been linked to the bmp biosynthetic gene cluster. This study explored the distribution and evolutionary history of this gene cluster in the genus Pseudoalteromonas. A phylogeny of the genus revealed numerous clades that do not contain type strains, suggesting considerable species level diversity has yet to be described. Comparative genomics revealed four distinct versions of the gene cluster distributed among 19 of the 101 Pseudoalteromonas genomes examined. These were largely localized to the least inclusive clades containing the Pseudoalteromonas luteoviolacea and Pseudoalteromonas phenolica type strains and show clear evidence of gene and gene cluster loss in certain lineages. Bmp gene phylogeny is largely congruent with the Pseudoalteromonas species phylogeny, suggesting vertical inheritance within the genus. However, the gene cluster is found in three different genomic environments suggesting either chromosomal rearrangement or multiple acquisition events. Bmp conservation within certain lineages suggests the encoded products are highly relevant to the ecology of these bacteria.

Dissemination of Genetic Acquisition/Loss Provides a Variety of Quorum Sensing Regulatory Properties in Pseudoalteromonas.

A bstract: Quorum sensing (QS) enables single-celled bacteria to communicate with chemical signals in order to synchronize group-level bacterial behavior. Pseudoalteromonas are marine bacteria found in versatile environments, of which QS regulation for their habitat adaptation is extremely fragmentary. To distinguish genes required for QS regulation in Pseudoalteromonas, comparative genomics was deployed to define the pan-genomics for twelve isolates and previously-sequenced genomes, of which acyl-homoserine lactone (AHL)-based QS traits were characterized. Additionally, transposon mutagenesis was used to identify the essential QS regulatory genes in the selected Pseudoalteromonas isolate. A remarkable feature showed that AHL-based colorization intensity of biosensors induced by Pseudoalteromonas most likely correlates with QS regulators genetic heterogeneity within the genus. This is supported by the relative expression levels of two of the main QS regulatory genes (luxO and rpoN) analyzed in representative Pseudoalteromonas isolates. Notably, comprehensive QS regulatory schema and the working model proposed in Pseudoalteromonas seem to phylogenetically include the network architectures derived from Escherichia coli, Pseudomonas, and Vibrio. Several associated genes were mapped by transposon mutagenesis. Among them, a right origin-binding protein-encoding gene (robp) was functionally identified as a positive QS regulatory gene. This gene lies on a genomic instable region and exists in the aforementioned bioinformatically recruited QS regulatory schema. The obtained data emphasize that the distinctly- and hierarchically-organized mechanisms probably target QS association in Pseudoalteromonas dynamic genomes, thus leading to bacterial ability to accommodate their adaption fitness and survival advantages.

Association of magnetotactic multicellular prokaryotes with Pseudoalteromonas species in a natural lagoon environment.

Magnetotactic bacteria, for the most part, are free-living, motile, unicellular prokaryotes that inhabit almost all marine and freshwater environments. One notable exception to the unicellular mode, however, are the magnetotactic multicellular prokaryotes. These morphologically unique prokaryotes (e.g., Candidatus Magnetoglobus multicellularis) are motile aggregates of 20-40 genetically identical, Gram-negative cells organised as a sphere (or ovoid in shape) and only motile as a unit. No specific close physical association between magnetotactic bacteria and non-magnetotactic microorganisms has ever been reported. Here, using culture-independent approaches, we show an unusual association between the spherical magnetotactic multicellular prokaryote Ca. Magnetoglobus multicellularis and Pseudoalteromonas species in environmental sediment and water samples collected from the Araruama Lagoon in Brazil. Cells of Pseudoalteromonas species were observed to be physically attached to the surface and, notably, even in the intercellular space of these spherical magnetotactic multicellular prokaryotes. An attempt to correlate the frequency of association between Pseudoalteromonas and magnetotactic multicellular prokaryotes with sediment depth was made but only a slight decrease in the number of Pseudoalteromonas cells per magnetotactic multicellular prokaryote was observed with increasing depth. Similar observations were made with magnetotactic multicellular prokaryotes from another Brazilian Lagoon (Rodrigo de Freitas) and the putative symbiont/parasite was detected. Although our results suggest some sort of specificity in the relationship between these prokaryotes, the precise nature of this association remains unclear.

Pseudoalteromonas piratica sp. nov., a budding, prosthecate bacterium from diseased Montipora capitata, and emended description of the genus Pseudoalteromonas.

A Gram-stain-negative, motile, rod-shaped bacterium designated OCN003T was cultivated from mucus taken from a diseased colony of the coral Montipora capitata in Kane'ohe Bay, O'ahu, Hawai'i. Colonies of OCN003T were pale yellow, 1-3 mm in diameter, convex, smooth and entire. The strain was heterotrophic, strictly aerobic and strictly halophilic. Cells of OCN003T produced buds on peritrichous prosthecae. Growth occurred within the pH range of 5.5 to 10, and the temperature range of 14 to 39 °C. Major fatty acids were 16 : 1ω7c, 16 : 0, 18 : 1ω7c, 17 : 1ω8c, 12 : 0 3-OH and 17 : 0. Phylogenetic analysis of 1399 nucleotides of the 16S rRNA gene nucleotide sequence and a multi-locus sequence analysis of three genes placed OCN003T in the genus Pseudoalteromonas and indicated that the nearest relatives described are Pseudoalteromonas spongiae, P. luteoviolacea, P. ruthenica and P. phenolica(97-99 % sequence identity). The DNA G+C content of the strain's genome was 40.0 mol%. Based on in silico DNA-DNA hybridization and phenotypic differences from related type strains, we propose that OCN003T represents the type strain of a novel species in the genus Pseudoalteromonas, proposed as Pseudoalteromonas piratica sp. nov. OCN003T (=CCOS1042T=CIP 111189T). An emended description of the genus Pseudoalteromonas is presented.

Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens.

Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented P. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of P. piscicida on lawns of the naturally occurring marine pathogens Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Photobacterium damselae, and Shewanella algae Inhibition also occurred on lawns of Staphylococcus aureus but not on Escherichia coli O157:H7 or Salmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three P. piscicida strains. Direct overlay of the Pseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of P. piscicida and a novel mechanism of direct transfer of these vesicles to V. parahaemolyticus Vesicles digested holes in V. parahaemolyticus cells, while the P. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which P. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCEPseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of P. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which P. piscicida kills V. parahaemolyticus It involves the direct transfer of apparently lytic vesicles from the surface of the Pseudoalteromonas strains to the surface of Vibrio cells, with subsequent digestion of holes in the Vibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of P. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.

ISCR2 is associated with the dissemination of multiple resistance genes among Vibrio spp. and Pseudoalteromonas spp. isolated from farmed fish.

58 multiresistant strains representing diverse genera were isolated from farmed fish in an aquaculture facility. Resistant rates of strains harboring ISCR2, an insertion sequence type element, were higher than those in which this element was absent. Full genome sequencing of a Vibrio isolate containing ISCR2 confirmed that it is associated with multiple resistance genes, many of which are of clinical relevance. We describe the structural variation within ISCR2, and its distribution throughout multiple diverse aquatic genera, including Vibrio, Shewenalla, Pseudoalteromonas and Psychrobacter, suggesting the potential role of ISCR2 in disseminating antibiotic resistance. We also observe, and experimentally verify, a novel macrolide resistance gene that is also associated with ISCR2.